It has long been recognized that certain polyunsaturated fatty acids, or PUFAs, are important biological components of healthy cells. For example, such PUFAs are recognized as:                “Essential” fatty acids that can not be synthesized de novo in mammals and instead must be obtained either in the diet or derived by further desaturation and elongation of linoleic acid (LA) or α-linolenic acid (ALA);        Constituents of plasma membranes of cells, where they may be found in such forms as phospholipids or triglycerides;        Necessary for proper development, particularly in the developing infant brain and for tissue formation and repair; and,        Precursors to several biologically active eicosanoids of importance in mammals, including prostacyclins, eicosanoids, leukotrienes and prostaglandins.        
In the 1970's, observations of Greenland Eskimos linked a low incidence of heart disease and a high intake of long-chain ω-3 PUFAs (Dyerberg, J. et al., Amer. J. Clin Nutr. 28:958-966 (1975); Dyerberg, J. et al., Lancet 2(8081):117-119 (Jul. 15, 1978)). More recent studies have confirmed the cardiovascular protective effects of ω-3 PUFAs (Shimokawa, H., World Rev Nutr Diet, 88:100-108 (2001); von Schacky, C., and Dyerberg, J., World Rev Nutr Diet, 88:90-99 (2001)). Further, it has been discovered that several disorders respond to treatment with ω-3 fatty acids, such as the rate of restenosis after angioplasty, symptoms of inflammation and rheumatoid arthritis, asthma, psoriasis and eczema. γ-linolenic acid (GLA, an ω-6 PUFA) has been shown to reduce increases in blood pressure associated with stress and to improve performance on arithmetic tests. GLA and dihomo-γ-linolenic acid (DGLA, another ω-6 PUFA) have been shown to inhibit platelet aggregation, cause vasodilation, lower cholesterol levels and inhibit proliferation of vessel wall smooth muscle and fibrous tissue (Brenner et al., Adv. Exp. Med. Biol. 83: 85-101 (1976)). Administration of GLA or DGLA, alone or in combination with eicosapentaenoic acid (EPA, an ω-3 PUFA), has been shown to reduce or prevent gastrointestinal bleeding and other side effects caused by non-steroidal anti-inflammatory drugs (U.S. Pat. No. 4,666,701). Further, GLA and DGLA have been shown to prevent or treat endometriosis and premenstrual syndrome (U.S. Pat. No. 4,758,592) and to treat myalgic encephalomyelitis and chronic fatigue after viral infections (U.S. Pat. No. 5,116,871). Other evidence indicates that PUFAs may be involved in the regulation of calcium metabolism, suggesting that they may be useful in the treatment or prevention of osteoporosis and kidney or urinary tract stones. Finally, PUFAs can be used in the treatment of cancer and diabetes (U.S. Pat. No. 4,826,877; Horrobin et al., Am. J. Clin. Nutr. 57 (Suppl.): 732S-737S (1993)).
PUFAs are generally divided into two major classes (consisting of the ω-6 and the ω-3 fatty acids) that are derived by desaturation and elongation of the essential fatty acids, LA and ALA, respectively (FIG. 1). Despite a variety of commercial sources of PUFAs from natural sources (e.g., seeds of evening primrose, borage and black currants; filamentous fungi (Mortierella), Porphyridium (red alga), fish oils and marine plankton (Cyclotella, Nitzschia, Crypthecodinium)), there are several disadvantages associated with these methods of production. First, natural sources such as fish and plants tend to have highly heterogeneous oil compositions. The oils obtained from these sources therefore can require extensive purification to separate or enrich one or more of the desired PUFAs. Natural sources are also subject to uncontrollable fluctuations in availability (e.g., due to weather, disease, or over-fishing in the case of fish stocks); and, crops that produce PUFAs often are not competitive economically with hybrid crops developed for food production. Large-scale fermentation of some organisms that naturally produce PUFAs (e.g., Porphyridium, Mortierella) can also be expensive and/or difficult to cultivate on a commercial scale.
As a result of the limitations described above, extensive work has been conducted toward: 1.) the development of recombinant sources of PUFAs that are easy to produce commercially; and 2.) modification of fatty acid biosynthetic pathways, to enable production of desired PUFAs. For example, advances in the isolation, cloning and manipulation of fatty acid desaturase and elongase genes from various organisms have been made over the last several years. Knowledge of these gene sequences offers the prospect of producing a desired fatty acid and/or fatty acid composition in novel host organisms that do not naturally produce PUFAs. The literature reports a number of examples in Saccharomyces cerevisiae, such as:    1. Domergue, F. et al. (Eur. J. Biochem. 269:4105-4113 (2002)), wherein two desaturases from the marine diatom Phaeodactylum tricornutum were cloned into S. cerevisiae, leading to the production of EPA;    2. Beaudoin F., et al. (Proc. Natl. Acad. Sci. U.S.A. 97(12):6421-6 (2000)), wherein the ω-3 and ω-6 PUFA biosynthetic pathways were reconstituted in S. cerevisiae, using genes from Caenorhabditis elegans;     3. Dyer, J. M. et al. (Appl. Eniv. Microbiol., 59:224-230 (2002)), wherein plant fatty acid desaturases (FAD2 and FAD3) were expressed in S. cerevisiae, leading to the production of ALA; and,    4. U.S. Pat. No. 6,136,574 (Knutzon et al., Abbott Laboratories), wherein one desaturase from Brassica napus and two desaturases from the fungus Mortierella alpina were cloned into S. cerevisiae, leading to the production of LA, GLA, ALA and STA.There remains a need, however, for an appropriate microbial system in which these types of genes can be expressed to provide for economical production of commercial quantities of one or more PUFAs. Additionally, a need exists for oils enriched in specific PUFAs, notably EPA and DHA.
One class or microorganisms that has not been previously examined as a production platform for PUFAs are the oleaginous yeasts. These organisms can accumulate oil up to 80% of their dry cell weight. The technology for growing oleaginous yeast with high oil content is well developed (for example, see EP 0 005 277 B1; Ratledge, C., Prog. Ind. Microbiol. 16:119-206 (1982)), and may offer a cost advantage compared to commercial micro-algae fermentation for production of ω-3 or ω-6 PUFAs. Whole yeast cells may also represent a convenient way of encapsulating ω-3 or ω-6 PUFA-enriched oils for use in functional foods and animal feed supplements.
Despite the advantages noted above, oleaginous yeast are naturally deficient in ω-6 and ω-3 PUFAs, since naturally produced PUFAs in these organisms are limited to 18:2 fatty acids (and less commonly, 18:3 fatty acids). Thus, the problem to be solved is to develop an oleaginous yeast that accumulates oils enriched in ω-3 and/or ω-6 fatty acids. Toward this end, it is necessary to introduce desaturases and elongases that allow for the synthesis and accumulation of ω-3 and/or ω-6 fatty acids in oleaginous yeasts. Despite availability of a variety of desaturase and elongase genes from numerous sources, these genes are not expressed with optimal efficiency in alternate hosts such as oleaginous yeast, since the codons in the genes do not reflect the typical codon usage of the alternate host organism. Thus, one must overcome problems associated with codon usage to optimize expression of PUFA genes in oleaginous yeast, to enable high-level production and accumulation of ω-3 and/or ω-6 fatty acids in these particular host organisms.
Applicants have solved the stated problem by developing means to codon-optimize desaturase and elongase genes suitable for expression in the oleaginous host, Yarrowia lipolytica. Exemplary genes optimized herein are those genes encoding a Δ6 desaturase, Δ17 desaturase and high affinity PUFA elongase, wherein codon-optimization improved the percent substrate conversion of LA to GLA (Δ6 desaturase) by approximately 40%, ARA to EPA by about 2-fold (Δ17 desaturase), and GLA to DGLA (elongase) by about 57% in Y. lipolytica. 